Volume 85, Issue 2, Pages 457-470 (January 2014) Expression patterns of RelA and c-mip are associated with different glomerular diseases following anti-VEGF therapy  Hassan Izzedine, Melanie Mangier, Virginie Ory, Shao-Yu Zhang, Kelhia Sendeyo, Khedidja Bouachi, Vincent Audard, Christine Péchoux, Jean C. Soria, Christophe Massard, Rastilav Bahleda, Edward Bourry, David Khayat, Alain Baumelou, Philippe Lang, Mario Ollero, Andre Pawlak, Djillali Sahali  Kidney International  Volume 85, Issue 2, Pages 457-470 (January 2014) DOI: 10.1038/ki.2013.344 Copyright © 2014 International Society of Nephrology Terms and Conditions

Figure 1 Differential expression of vascular endothelial growth factor (VEGF), hypoxia-inducible factor 1α (HIF-1α), c-mip, RelA, and Tie2 in glomeruli from patients with minimal change nephropathy/focal and segmental glomerulopathy (MCN/FSG)–like lesions or thrombotic microangiopathy (TMA) following anti-VEGF or receptor tyrosine kinase inhibitor (RTKI) therapies. Representative immunohistochemical analysis of VEGF (a), c-mip (b), HIF-1α (c), RelA (d), and Tie2 (e) in kidney biopsies from patients with renal diseases associated with anti-VEGF drugs. Note that VEGF is not detected in TMA, whereas it is little reduced in MCN/FSG-like lesions. c-mip is not detected in the glomeruli of control human kidneys (Con) or in TMA, whereas it is clearly visualized along the external side of the glomerular capillary loops in MCN. The expression of HIF-1α is highly increased in TMA glomeruli, whereas it is scarcely detectable in MCN/FSG-like lesions. Within TMA glomeruli, HIF-1α is mostly restricted to podocyte nuclei. The abundance of RelA is strongly increased in a segmental pattern in TMA glomeruli, whereas it is decreased in MCN/FSG-like lesions, as compared with control glomeruli (Con). Tie2 was significantly upregulated after anti-VEGF or RTKI therapies. Bars = 20μm. The relative abundance of these markers was measured by computer-assisted image analysis using × 400 magnification and is shown in f. iMCNS, idiopathic MCN syndrome; iFSGS, idiopathic focal and segmental glomerulosclerosis; TMAvegf, TMA induced by VEGF; iTMA, idiopathic TMA. Kidney International 2014 85, 457-470DOI: (10.1038/ki.2013.344) Copyright © 2014 International Society of Nephrology Terms and Conditions

Figure 2 Differential expression of RelA and nephrin in thrombotic microangiopathy (TMA) and minimal change nephropathy (MCN)-like lesions. Confocal microscopy analysis of nephrin (red) and RelA (green) expression in control human kidneys (Con), MCN-like lesions (MCN), and TMA biopsies. The abundance of RelA is significantly increased in TMA. RelA is colocalized with nephrin, but it is also abundant on the inside of capillary loops, within the TMA glomeruli. Bars = 10μm. Kidney International 2014 85, 457-470DOI: (10.1038/ki.2013.344) Copyright © 2014 International Society of Nephrology Terms and Conditions

Figure 3 Differential expression of RelA and Tie2 in thrombotic microangiopathy (TMA) and minimal change nephropathy (MCN)-like lesions. Confocal microscopy analysis of Tie2 (red) and RelA (green) expression in control glomeruli (Con), MCN-like lesions (MCN), and TMA biopsies. The abundance of RelA and Tie2 is significantly increased in TMA. Tie2 and RelA are colocalized in damaged areas within TMA glomeruli, whereas they are expressed in different cell compartments in control glomeruli. Bars = 10μm. Kidney International 2014 85, 457-470DOI: (10.1038/ki.2013.344) Copyright © 2014 International Society of Nephrology Terms and Conditions

Figure 4 Overproduction of RelA in endothelials cells in thrombotic microangiopathy (TMA). Confocal microscopy analysis of Tie2 (red) and RelA (green) expression in arterioles of control human kidneys (Con) and TMA biopsies. The abundance of RelA and Tie2 is significantly increased in TMA arterioles, which display a swelling of endothelial cells. The abundance of RelA is also increased in the pericytes of TMA arterioles. Bars = 10μm. The relative abundance of RelA was assessed by quantifying the specific arteriolar fluorescence intensity in three-dimensional stacks of images taken by confocal microscopy and normalized to total arteriolar area. Five samples were analyzed in each condition (Con and TMA). Data represent the mean±s.e.m. (*P<0.05, Mann–Whitney test). Kidney International 2014 85, 457-470DOI: (10.1038/ki.2013.344) Copyright © 2014 International Society of Nephrology Terms and Conditions

Figure 5 Electron microscopy analysis and distribution of RelA by immunogold labeling in thrombotic microangiopathy (TMA). (a) Representative transmission electron micrographs of the glomeruli in a patient with bevacizumab-induced TMA (right panel) and in a control kidney (left panel). (b) Representative immunogold labeling for Rel A (10nm gold, indicated by the asterisks) in the glomeruli of a patient with bevacizumab-induced TMA (right panel) and in a control kidney (left panel). (c) Quantification of gold label for RelA in glomeruli patients with TMA (n=3) and in control (n=3) (NS, nonsignificant, P=0.6993; **P=0.0026; ***P=0.0006, Mann–Whitney test). CL, capillary lumen; E, endothelium; EC, endothelial cells; FP, foot process; GBM, glomerular basement membrane; P, podocyte. Kidney International 2014 85, 457-470DOI: (10.1038/ki.2013.344) Copyright © 2014 International Society of Nephrology Terms and Conditions

Figure 6 RelA binds to the c-mip promoter and prevents its transcriptional activity. (a) Characterization of the c-mip promoter. Nucleotide sequence of the human c-mip proximal promoter region. Nucleotides are numbered to the left of the sequence with the transcription start site (TSS) indicated by the +1 position. The potential CCAAT site and nuclear factor-κB (NF-κB) responsive element (NF-κB RE) are indicated. (b) Identification of NF-κB RE by chromatin immunoprecipitation (IP). HEK cells were cross-linked with formaldehyde, and chromatin was immunoprecipitated with antibodies against either rabbit polyclonal anti-RelA antibody or rabbit IgG (control). Immunoprecipitated DNA was analyzed by PCR using primers flanking the NF-κB RE within the human promoter (the region amplified spans from -279 to -72 relative to the transcriptional start site). (c) RelA binds to NF-κB recognition site on the c-mip promoter. HEK cells were transiently cotransfected with either RelA expression plasmid or its empty vector (Ev). Protein extracts (20μg) were used for NF-κB DNA-binding assays. Nuclear extracts were incubated with the wild-type NF-κB oligonucleotide in the absence or presence of anti-RelA/p65 antibody. The specificity of NF-κB interaction was monitored by using a mutant NF-κB oligonucleotide. The quantification of NF-κB band shifts is indicated. (d) RelA inhibits the c-mip promoter-dependent luciferase activity. HEK cells were cotransfected with NF-κB (RelA/p65, p50) expression plasmids and the human c-mip-luciferase reporter plasmid (pGl3-C-mip-luc). The phRL-null vector was used as an internal control for transfection. Cell extracts were prepared 24h after transfection, and then luciferase activity was measured and normalized by protein content determined by using a Bradford assay. Data are presented as relative luciferase activity (firefly luciferase/Renilla luciferase). The two-tailed Student’s t-test is used for statistical analysis (***P<0.001, Mann–Whitney test). Five independent experiments were performed. Kidney International 2014 85, 457-470DOI: (10.1038/ki.2013.344) Copyright © 2014 International Society of Nephrology Terms and Conditions

Figure 7 Overproduction of c-mip in RelA-deficient cells. Relative expression of RelA and c-mip transcripts on total RNA from wild-type and RelA-deficient mouse embryonic fibroblast (MEF). Bars represent the mean value of five independent experiments with error bars indicating s.e.m. (***P<0.001, Mann–Whitney test). Kidney International 2014 85, 457-470DOI: (10.1038/ki.2013.344) Copyright © 2014 International Society of Nephrology Terms and Conditions

Figure 8 Receptor tyrosine kinase inhibitor (RTKI) sorafenib induces upregulation of c-mip and a profound cytoskeleton disorganization. (a) Left, western blot analysis of c-mip on total protein lysates from wild-type and RelA-deficient mouse embryonic fibroblasts (MEFs), with or without treatment with sorafenib (10μmol/l). The membrane was reblotted with glyceraldehydes 3-phosphate dehydrogenase (GAPDH) antibody; right, relative abundance of c-mip corrected by GAPDH signal (from left panel). Similar results were obtained in two independent experiments. (b) Relative expression of c-mip transcript on total RNA from differentiated podocytes either nontreated or treated with sorafenib (10μmol/l). Bars represent the mean value of three independent experiments with error bars indicating s.e.m. (*P<0.05, Mann–Whitney test). (c) Sorafenib induces stress-fiber depletion with abnormal production of enclosed F-actin-rich membrane structures in podocytes. Differentiated podocytes were incubated with either 10μmol/l sorafenib or the vehicle (control) for 24h at 37°C, and then stained for F-actin with fluorescein isothiocyanate–conjugated phalloidin. Control podocytes (Con) display normal cell morphology with multiple arborizing cell processes, whereas podocytes exposed to sorafenib lose their normal shape, become elongated, and lose the contact with neighboring cells through their processes. Sorafenib causes significant loss of stress fibers, whereas many full F-actin rings are newly formed in the cytoplasm. (d) Western blot analysis of c-mip on total protein lysates from normal lymphocytes with (+) or without (-) treatment with sorafenib (10μmol/l). Kidney International 2014 85, 457-470DOI: (10.1038/ki.2013.344) Copyright © 2014 International Society of Nephrology Terms and Conditions

Figure 9 Sorafenib inhibits RelA activation. (a) Western blot analysis of pSer32/36 IκBα and pSer276 RelA on total protein lysates from podocytes, with (+) or without (-) treatment with sorafenib (10μmol/l). The quantification of phosphorylated forms on total proteins is indicated in the lower panels. Bars represent the mean value of three independent experiments, with error bars indicating s.e.m. (*P<0.05, one-way analysis of variance test). (b) Western blot analysis of RelA on cytoplasm and nuclear podocyte extracts, of which the purity was assessed by calpain and Sp1 blotting, respectively. Data are representative of three independent experiments. (c) Immunoprecipitation of RelA (rabbit antibody) from podocyte protein lysates, followed by sequential immunoblotting with mouse IκBα and RelA antibodies. (d) Confocal microscopy analysis of cells with or without treatment with sorafenib (10μmol/l) for 48h. Kidney International 2014 85, 457-470DOI: (10.1038/ki.2013.344) Copyright © 2014 International Society of Nephrology Terms and Conditions